PROJECTSUMMARY Smallcelllungcancer(SCLC)isthemostaggressiveformoflungcancer,withamediansurvivalof24 monthsandafive-yearoverallsurvivalof7%.Cisplatin-basedchemotherapyisthefirst-linetreatmentforSCLC, functioning by creating DNA crosslinks leading to cancer cell death. While initially effective, many patients ultimatelydevelopcisplatinresistanceandexperiencetumorrecurrence.TheDNAdamageresponse(DDR)is asignalingnetworkthatrecognizeschallengestogenomeintegrityandcoordinatesdiverseDNArepairandcell cycle checkpoint pathways. Targeting DDR proteins critical to the cellular response to cisplatin in SCLC may overcome acquired cisplatin resistance, but the cellular response to cisplatin remains unclear because many proteins that respond to cisplatin-induced DNA damage have yet to be identified. To address this issue, a syntheticlethalscreenusingacustomsiRNAlibraryof1008nuclearenzymeswasperformedinH128cisplatin- resistantSCLCcellstoidentifygenescriticalformediatingcisplatinresistance.EnhancerOfZeste2Polycomb Repressive Complex 2 Subunit (EZH2), a H3 K27 methyltransferase, was identified as one of the strongest syntheticlethalhitsinthescreen,whereknockdownofEZH2stronglysensitizedH128cellstocisplatin. EZH2isoverexpressedinSCLCaswellasothercancersandhasbeenimplicatedincancerprogression. Moreover,EZH2inhibitorshaveshownefficacyinseveralcancers,includingSCLC;?however,thethemolecular underpinnings of the efficacy of EZH2 depletion or inhibition in SCLC and the mechanistic role of EZH2 in mediatingcisplatinresistanceinSCLCareunclear.Inthisregard,IvalidatedthatEZH2depletionincisplatin- resistantH128andH146SCLCcelllinesresultsincisplatinhypersensitivity,andfurthermorefoundthatEZH2 localizestoDNAdamagesitesinducedbylasermicroirradiation,suggestingthatEZH2mayfunctiondirectlyin mediating DNA damage resistance in SCLC. In addition, using mass spectrometry analysis of purified EZH2 fromcells,IidentifiedanovelinteractionbetweenEZH2andDNADamage-BindingProtein1(DDB1),anE3 ubiquitinligasecomponentthatpromotesnucleotideexcisionrepair(NER)ofcisplatin-inducedDNAintrastrand crosslinks,whichIvalidatedbyco-immunoprecipitation.Assuch,IhypothesizethatEZH2playsacriticalrolein mediatingcisplatinresistanceinSCLCbypromotingDNAdamageresponse(DDR)activitiesandfurthermore thatEZH2inhibitionwillsensitizeresistantSCLCcellsandtumorswithdysregulatedDDRpathwaystocisplatin treatment.Aim1willdeterminethemechanismbywhichEZH2mediatescisplatinresistanceinSCLC.Aim2will determine if EZH2 inhibition sensitizes resistant SCLC cells and tumors with dysregulated DDR protein expressiontocisplatintreatmentinvitroandinvivo.CompletionoftheseaimswilldefineanovelroleforEZH2 in the DDR in mediating cisplatin resistance in SCLC and elucidate the effectiveness and specific biological context for which EZH2 can function as a novel therapeutic target for improving the efficacy of treatment for patientswithSCLCwhofailinitialtreatment.